About

Mark P. Brynildsen is a Professor of Chemical and Biological Engineering at Princeton University and is affiliated with the Omenn-Darling Bioengineering Institute. His research focuses on addressing the critical public health issue of antibiotic resistance by employing experimental and computational techniques in systems biology and synthetic biology to understand and combat infectious diseases. His main areas of investigation include bacterial persistence, a non-genetic ability of bacteria to tolerate antibiotics, and bacterial defenses against immune antimicrobials. His work aims to identify physiological elements that enable bacteria to survive antibiotic treatments and to develop novel treatments based on this understanding. Additionally, Brynildsen explores antivirulence therapies that disrupt essential host-pathogen interactions, thereby increasing bacterial susceptibility to immune attack and reducing the likelihood of resistance development. His contributions have advanced knowledge in bacterial stress responses, metabolism, and persistence, and he has been recognized with numerous awards for his research, mentorship, and service.

Research topics

• Genetics
• Biology
• Microbiology
• Cell biology

Selected publications

• Conservation and divergence of ciprofloxacin persister survival mechanisms between Pseudomonas aeruginosa and Escherichia coli

  PLoS Genetics · 2025-09-02 · 1 citations

  articleOpen accessSenior authorCorresponding

  Studies have shown that DNA damage repair systems, including homologous recombination (HR) and the SOS response, are important for fluoroquinolone (FQ) persistence of Escherichia coli, which has been the workhorse organism of persister research. We sought to explore whether those systems are also important for FQ persistence of Pseudomonas aeruginosa, a common cause of lung infections in cystic fibrosis patients, which can be treated with FQs such as ciprofloxacin (CIP). Notably, P. aeruginosa has important differences in its DNA damage repair capabilities compared to E. coli that include the machinery needed to conduct non-homologous end-joining (NHEJ), Ku and LigD. Using a genetic approach, we found that loss of HR significantly depressed persister levels of P. aeruginosa to CIP during stationary-phase, but not in exponential-phase. This differed from E. coli grown in identical conditions, where loss of HR reduced survival in both stationary- and exponential-phase populations. Similarly, an inability to induce the SOS response reduced survival during both growth phases for E. coli but only in stationary-phase for P. aeruginosa. Loss of NHEJ machinery in P. aeruginosa did not impact persister levels during stationary- or exponential-phase, whereas overexpression of NHEJ machinery in P. aeruginosa had toxic effects. In addition, the generality of findings to another FQ, levofloxacin, and a recent clinical isolate, MRSN 1612, were confirmed. These results demonstrate that HR and the SOS response are important to CIP persistence of stationary-phase P. aeruginosa, dispensable to CIP persisters in growing P. aeruginosa cultures, and that the contributions of systems to E. coli persistence do not directly translate to persisters of P. aeruginosa.

  PublisherOA PDFDOI

• Solidification of protein aggregates deepens bacterial dormancy

  Trends in Microbiology · 2025-03-19 · 2 citations

  articleOpen accessSenior author
  PublisherOA PDFDOI

• An investigation of gene dosage reveals that increased sensitivity to D-cycloserine divergently impacts the transience of heteroresistance in <i>Escherichia coli</i>

  mBio · 2025-08-22

  articleOpen accessSenior author

  ABSTRACT Heteroresistance describes the phenomenon where seemingly isogenic bacterial populations contain subpopulations with elevated resistance compared to the susceptible majority that are often missed in routine susceptibility testing. The enhanced resistance of those subpopulations can either be maintained (stable) or quickly lost (unstable/transient) after treatment, where transient cases return susceptibility results identical to those of original cultures. Recent work has implicated increased gene dosage of resistance determinants as a major cause of unstable heteroresistance in clinical isolates. Inspired by that work, we sought to systematically evaluate how gene dosage of an antibiotic’s target network components impacted heteroresistance and its stability. To accomplish that, we used Escherichia coli MG1655 as a model organism, and D-cycloserine (DCS), a cell-wall synthesis inhibitor that enters through a transporter (CycA) and inhibits multiple enzymes (DdlA, DdlB, DadX, and Alr), as a model antibiotic. To measure heteroresistance, we used population analysis profiling, and to quantify stability, we used a population-level model to define a heteroresistance stability index ( <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" display="inline" overflow="scroll"> <mml:mstyle displaystyle="true" scriptlevel="0"> <mml:mrow> <mml:mrow> <mml:msub> <mml:mrow> <mml:mi mathvariant="normal">H</mml:mi> <mml:mi mathvariant="normal">S</mml:mi> <mml:mi mathvariant="normal">I</mml:mi> </mml:mrow> <mml:mi mathvariant="normal">j</mml:mi> </mml:msub> </mml:mrow> <mml:msub> <mml:mo stretchy="false">∣</mml:mo> <mml:mi>t</mml:mi> </mml:msub> </mml:mrow> </mml:mstyle> </mml:math> ), which quantifies the proportion of heteroresistant subpopulation j that maintained elevated resistance over t generations. We found that increased sensitivity to DCS through gene dosage variation of cycA and ddlB divergently impacted heteroresistance stability, with ddlB enhancing stability and cycA fostering transience. These findings translated to uropathogenic E. coli (UTI89) and suggested that increasing the number of antibiotic targets and/or points of antibiotic entry could decrease the propensity of heteroresistance to yield stable resistance. This knowledge could impact the development of new antibiotics and improve understanding of antibiotic treatment failure. IMPORTANCE Heteroresistance is a concern because heteroresistant strains escape clinical detection and facilitate treatment failure. Heteroresistant cells can produce stably resistant or transiently resistant populations, and enhanced understanding of genetic factors that influence the level of heteroresistance and its stability has the potential to improve treatment strategies. Here, we introduce the heteroresistance stability index, which is a quantitative metric of heteroresistance stability, and use it to analyze heteroresistance of Escherichia coli to D-cycloserine. We investigated how gene dosage of antibiotic target network components (transporter, enzymatic targets) influences heteroresistance and its stability and found diverging outcomes on stability for comparable declines in heteroresistance. Specifically, these results suggest that designing antibiotics to enter through multiple transporters or target multiple enzymes would reduce the emergence of stable resistance.

  PublisherDOI

• Loss of Gre factors leads to phenotypic heterogeneity and cheating in <i>Escherichia coli</i> populations under nitric oxide stress

  mBio · 2024-09-09 · 2 citations

  articleOpen accessSenior author

  ABSTRACT Nitric oxide (·NO) is one of the toxic metabolites that bacteria can be exposed to within phagosomes. Gre factors, which are also known as transcript cleavage factors or transcription elongation factors, relieve back-tracked transcription elongation complexes by cleaving nascent RNAs, which allows transcription to resume after stalling. Here we discovered that loss of both Gre factors in Escherichia coli , GreA and GreB, significantly compromised ·NO detoxification due to ·NO-induced phenotypic heterogeneity in Δ greA Δ greB populations, which did not occur in wild-type cultures. Under normal culturing conditions, both wild-type and Δ greA Δ greB synthesized transcripts uniformly, whereas treatment with ·NO led to bimodal transcript levels in Δ greA Δ greB that were unimodal in wild-type. Interestingly, exposure to another toxic metabolite of phagosomes, hydrogen peroxide (H 2 O 2 ), produced analogous results. Furthermore, we showed that loss of Gre factors led to cheating under ·NO stress where transcriptionally deficient cells benefited from the detoxification activities of the transcriptionally proficient subpopulation. Collectively, these results show that loss of Gre factor activities produces phenotypic heterogeneity under ·NO and H 2 O 2 stress that can yield cheating between subpopulations. IMPORTANCE Toxic metabolite stress occurs in a broad range of contexts that are important to human health, microbial ecology, and biotechnology, whereas Gre factors are highly conserved throughout the bacterial kingdom. Here we discovered that loss of Gre factors in E. coli leads to phenotypic heterogeneity under ·NO and H 2 O 2 stress, which we further show with ·NO results in cheating between subpopulations. Collectively, these data suggest that Gre factors play a role in coping with toxic metabolite stress, and that loss of Gre factors can produce cheating between neighbors.

  PublisherDOI

• Differential impacts of DNA repair machinery on fluoroquinolone persisters with different chromosome abundances

  mBio · 2024-04-02 · 6 citations

  articleOpen accessSenior author

  ABSTRACT DNA repair machinery has been found to be indispensable for fluoroquinolone (FQ) persistence of Escherichia coli . Previously, we found that cells harboring two copies of the chromosome (2Chr) in stationary-phase cultures were more likely to yield FQ persisters than those with one copy of the chromosome (1Chr). Furthermore, we found that RecA and RecB were required to observe that difference, and that loss of either more significantly impacted 2Chr persisters than 1Chr persisters. To better understand the survival mechanisms of persisters with different chromosome abundances, we examined their dependencies on different DNA repair proteins. Here, we show that lexA3 and ∆ recN negatively impact the abundances of 2Chr persisters to FQs, without significant impacts on 1Chr persisters. In comparison, ∆ xseA , ∆ xseB , and ∆ uvrD preferentially depress 1Chr persistence to levels that were near the limit of detection. Collectively, these data show that the DNA repair mechanisms used by persisters vary based on chromosome number, and suggest that efforts to eradicate FQ persisters will likely have to take heterogeneity in single-cell chromosome abundance into consideration. IMPORTANCE Persisters are rare phenotypic variants in isogenic populations that survive antibiotic treatments that kill the other cells present. Evidence has accumulated that supports a role for persisters in chronic and recurrent infections. Here, we explore how an under-appreciated phenotypic variable, chromosome copy number (#Chr), influences the DNA repair systems persisters use to survive fluoroquinolone treatments. We found that #Chr significantly biases the DNA repair systems used by persisters, which suggests that #Chr heterogeneity should be considered when devising strategies to eradicate these troublesome bacterial variants.

  PublisherDOI

• Amino acids can deplete ATP and impair nitric oxide detoxification by Escherichia coli

  Free Radical Biology and Medicine · 2023-05-28 · 1 citations

  articleSenior authorCorresponding
  PublisherDOI

• High-Throughput Screen Reveals the Structure–Activity Relationship of the Antimicrobial Lasso Peptide Ubonodin

  ACS Central Science · 2023-03-01 · 31 citations

  articleOpen access

  . Ubonodin and several of the variants identified in this study had lower MICs against certain Bcc strains than those of many clinically approved antibiotics. Finally, the large library size enabled us to develop DeepLasso, a deep learning model that can predict the RNAP inhibitory activity of an ubonodin variant.

  PublisherOA PDFDOI

• Gre factors protect against phenotypic diversification and cheating in <i>Escherichia coli</i> populations under toxic metabolite stress

  bioRxiv (Cold Spring Harbor Laboratory) · 2023-01-03

  preprintOpen accessSenior authorCorresponding

  Abstract Nitric oxide (·NO) is one of the toxic metabolites that bacteria can be exposed to within phagosomes. Gre factors, which are also known as transcript cleavage factors or transcription elongation factors, relieve back-tracked transcription elongation complexes by cleaving nascent RNAs, which allows transcription to resume after stalling. Here we discovered that loss of both Gre factors in E. coli , GreA and GreB, significantly compromised ·NO detoxification through a phenotypic diversification of the population. Under normal culturing conditions, both wild-type and Δ greA Δ greB synthesized protein uniformly. However, treatment with ·NO led to bimodal protein expression in Δ greA Δ greB , whereas wild-type remained unimodal. Interestingly, exposure to another toxic metabolite of phagosomes, hydrogen peroxide (H 2 O 2 ), produced similar results. We found that the diversification in Δ greA Δ greB cultures required E. coli RNAP, occurred at the level of transcription, and could produce cheating where transcriptionally-deficient cells benefit from the detoxification activities of the transcriptionally-proficient subpopulation. Collectively, these results indicate that Gre factors bolster bacterial defenses by preventing phenotypic diversification and cheating in environments with fast-diffusing toxic metabolites. Importance Toxic metabolite stress occurs in a broad range of contexts that are important to human health, microbial ecology, and biotechnology; whereas Gre factors are highly conserved throughout the bacterial kingdom. Here we discovered that the Gre factors of E. coli prevent phenotypic diversification under toxic metabolite stress. Such conformist regulation improves populationwide removal of those stressors and protects against cheating, where one subpopulation commits resources to counter a threat, and the other subpopulation does not, yet both subpopulations benefit.

  PublisherOA PDFDOI

• Cephalosporin resistance, tolerance, and approaches to improve their activities

  The Journal of Antibiotics · 2023-12-19 · 25 citations

  articleSenior author
  PublisherDOI

• Genome-wide mapping of fluoroquinolone-stabilized DNA gyrase cleavage sites displays drug specific effects that correlate with bacterial persistence

  Nucleic Acids Research · 2022-12-12 · 12 citations

  articleOpen accessSenior authorCorresponding

  Bacterial persisters are rare phenotypic variants that are suspected to be culprits of recurrent infections. Fluoroquinolones (FQs) are a class of antibiotics that facilitate bacterial killing by stabilizing bacterial type II topoisomerases when they are in a complex with cleaved DNA. In Escherichia coli, DNA gyrase is the primary FQ target, and previous work has demonstrated that persisters are not spared from FQ-induced DNA damage. Since DNA gyrase cleavage sites (GCSs) largely govern the sites of DNA damage from FQ treatment, we hypothesized that GCS characteristics (e.g. number, strength, location) may influence persistence. To test this hypothesis, we measured genome-wide GCS distributions after treatment with a panel of FQs in stationary-phase cultures. We found drug-specific effects on the GCS distribution and discovered a strong negative correlation between the genomic cleavage strength and FQ persister levels. Further experiments and analyses suggested that persistence was unlikely to be governed by cleavage to individual sites, but rather survival was a function of the genomic GCS distribution. Together, these findings demonstrate FQ-specific differences in GCS distribution that correlate with persister levels and suggest that FQs that better stabilize DNA gyrase in cleaved complexes with DNA will lead to lower levels of persistence.

  PublisherOA PDFDOI

Recent grants

• Examining fluoroquinolone-induced DNA damage in persisters and its contributions to antibiotic resistance.

  NIH · $2.0M · 2017–2023

• CAREER: Metabolic Engineering to Potentiate Immunity and Discover Novel Antivirulence Therapies

  NSF · $500k · 2015–2020

• Exploring Persister Antibiotic Responses as a Source of Biomarkers and Elimination Strategies

  NIH · $446k · 2015–2018

• High-throughput Quantification of Persister Physiology at the Systems Level

  NIH · $430k · 2016–2019

Frequent coauthors

• James J. Collins

  Massachusetts Institute of Technology

  13 shared

• James C. Liao

  Institute of Biological Chemistry, Academia Sinica

  12 shared

• Mehmet A. Orman

  12 shared

• Theresa C. Henry

  Rutgers, The State University of New Jersey

  11 shared

• Allison M. Murawski

  University of Florida

  10 shared

• Wendy W. K. Mok

  UConn Health

  10 shared

• Darshan M. Sivaloganathan

  Princeton University

  9 shared

• Jonathan L. Robinson

  Chalmers University of Technology

  9 shared

Awards & honors

• Distinguished Service Award, AIChE Food, Pharmaceutical and…
• Graduate Mentoring Award, McGraw Center for Teaching and Lea…
• NJ Health Foundation Award, 2021
• 250th Anniversary Fund for Innovation in Undergraduate Educa…
• Princeton Engineering Council Excellence in Teaching Award,…